We have determined that hMOF, the human ortholog of the Drosophila MOF gene (males absent on the first), encoding a protein with histone acetyltransferase activity, interacts with the ATM (ataxia-telangiectasiamutated) protein. Cellular exposure to ionizing radiation (IR) enhances hMOF-dependent acetylation of its target substrate, lysine 16 (K16) of histone H4 independently of ATM function. Blocking the IR-induced increase in acetylation of histone H4 at K16, either by the expression of a dominant negative mutant ⌬hMOF or by RNA interference-mediated hMOF knockdown, resulted in decreased ATM autophosphorylation, ATM kinase activity, and the phosphorylation of downstream effectors of ATM and DNA repair while increasing cell killing. In addition, decreased hMOF activity was associated with loss of the cell cycle checkpoint response to DNA double-strand breaks. The overexpression of wild-type hMOF yielded the opposite results, i.e., a modest increase in cell survival and enhanced DNA repair after IR exposure. These results suggest that hMOF influences the function of ATM.In eukaryotic cells, DNA damage activates signal transduction pathways that rapidly affect downstream processes such as gene transcription, cell cycle progression, and DNA replication (13,25). All of these processes involve alterations in chromatin structure. Posttranslational covalent modifications of histones have emerged as key regulatory events in DNA damage response. A widespread modification is acetylation catalyzed by histone acetyltransferases and reversed by deacetylases (3, 13, 50). Reversible acetylation of four lysines (K) at positions 5, 8, 12, and 16 in the amino-terminal tail of histone H4 occurs in vivo in all eukaryotes (3). The hyperacetylation of histone H4 could lead to the unfolding of the nucleosomal fiber (50), and the acetylation of histone H4 at K16 occurs on the hyperactive male X chromosome of Drosophila polytene chromosomes (51). Ikura et al. (19) noted that Tip60 (Tat-interacting protein), which acetylates histones H2A, H3, and H4, plays a role in DNA repair. More recently, Kusch et al. (28) demonstrated that the Drosophila Tip60 acetylates nucleosomal phosphoH2Av and exchanges it with an unmodified H2Av. Bird et al. (5) reported that the acetylation of histone H4 by Esa1 (essential SAS2-related acetyltransferase) is required for DNA repair in yeast and suggested that a similar modification may function in mammalian cells.ATM (ataxia-telangiectasia-mutated protein) is crucial for the initiation of signaling pathways in mammalian cells following exposure to ionizing radiation (IR) and other DNA-damaging agents (36, 46), and cells deficient in ATM function also have defective telomere chromatin (47). Bakkenist and Kastan (4) have suggested that chromatin structural perturbations induced by DNA double-strand breaks (DSBs) serve as a trigger for ATM activation. Recent studies indicate that the MRN (Mre11, Rad50, and Nbs1) complex as well as TRF2 either influences activation of ATM (9, 29, 52) or serves as a modulator/amp...

The mammalian ortholog of the Drosophila MOF (males absent on the first) gene product is a histone H4 lysine 16-specific acetyltransferase. Recent studies have shown that depletion of human MOF (hMOF) in human cell lines leads to genomic instability, spontaneous chromosomal aberrations, cell cycle defects, altered nuclear morphology, reduced transcription of certain genes, and defective DNA damage response to ionizing radiation (IR). Here we show that MOF plays an essential role in mammals during embryogenesis and oncogenesis. Ablation of the mouse Mof gene (mMof) by gene targeting resulted in early embryonic lethality and cell death. Lethality correlated with the loss of H4 lysine 16 acetylation (H4K16ac) and could not be rescued by concomitant inactivation of ATM or p53. In comparison to primary cells or normal tissue, all immortalized human normal and tumor cell lines and primary tumors demonstrated similar or elevated hMOF and H4K16ac levels. Accordingly, MOF overexpression correlated with increased cellular proliferation, oncogenic transformation, and tumor growth. Thus, these data reveal that the acetylation of histone H4 at K16 by MOF is an epigenetic signature of cellular proliferation common to both embryogenesis and oncogenesis and that MOF is an essential factor for embryogenesis and oncogenesis.MOF belongs to the MYST family of acetyltransferases, which have been associated with acute myeloid leukemia (MOZ), transcriptional silencing in Saccharomyces cerevisiae (SAS2 and YBF2/SAS3), interactions with human immunodeficiency virus Tat in humans (TIP60), and dosage compensation in Drosophila melanogaster (MOF), in addition to their role in DNA damage repair (2,4,12,13,17,18,43,44,46). Akhtar and Becker (1) demonstrated that Drosophila MOF is a histone acetyltransferase that acetylates chromatin specifically at histone H4 lysine 16 (H4K16). Depletion of human MOF (hMOF) in human cells results in the loss of acetylation at lysine 16 of histone H4 (6, 12, 41, 45, 46), strongly arguing that the highly conserved MOF protein may be the major histone acetyltransferase, which acetylates histone H4 at K16.Acetylation at K16 of histone H4 (H4K16ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes, and recent studies have highlighted its significance. Shogren-Knaak and coworkers have found that a single histone H4K16ac modification modulates both higher-order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber (39). Shia and coworkers have demonstrated that the presence of H4K16ac and H2A.Z synergistically prevent the ectopic propagation of heterochromatin in the subtelomeric regions of yeast (36). Furthermore, it is well understood that H4K16ac disrupts higher-order chromatin structure, changes the functional interactions between chromatin-associated proteins (39), and serves as a switch for altering chromatin from a repressive to a transcriptionally active state in yeast and humans (36). Interestingly, Dou and coworkers reported...

All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (;-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces ;-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced ;-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IRinduced signaling required for chromosomal DNA doublestrand break repair, thus resulting in increased cellular radiosensitivity. [Cancer Res 2007;67(7):3010-7]

We tested the hypothesis that manganese superoxide dismutase (MnSOD), an antioxidant enzyme, regulates the proliferative potential of confluent human fibroblasts. Normal human skin (AG01522) and lung (WI38, CCL-75) fibroblasts kept in confluence (>95% G 0 /G 1 ) showed a significant decrease in their capacity to reenter the proliferation cycle after 40 -60 days. The inhibition of re-entry was accompanied with the agedependent increase of p16 protein levels in the confluent culture. Adenoviral mediated overexpression of MnSOD during confluent growth suppressed p16, enhanced p21 protein accumulation, and protected fibroblasts against the loss of proliferation potential. Increases in p21 protein levels in MnSOD overexpressing confluent fibroblasts were independent of p53 protein levels. p53 protein levels did not change in control, replication-defective adenovirus containing an insertless vector (AdBgl II), or AdMnSOD-infected confluent cells cultured for 20 and 60 days. In addition, MnSOD-induced protection of the proliferation capacity of confluent fibroblasts was independent of their telomerase activity. However, telomerase-transformed fibroblasts showed increased MnSOD expression in confluent growth, maintaining their capacity to re-enter the proliferation cycle. Although inactivation of the retinoblastoma protein in cells subcultured from the 60-day confluent control, AdBgl II-, and AdMnSOD-infected fibroblasts was identical, only MnSOD-overexpressing cells showed a higher percentage of S-phase. These results support the hypothesis that a redox-sensitive checkpoint regulated the progression of fibroblasts from G 0 /G 1 to S-phase.In mammalian cells, intracellular antioxidant enzymes include superoxide dismutase, catalase, and glutathione peroxidase. There are two intracellular forms of superoxide dismutase as follows: CuZnSOD, 1 found in the cytoplasm and nucleus, and MnSOD, found in mitochondria (1, 2). Different isozymes of glutathione peroxidase are found in most subcellular compartments, whereas catalase is found primarily in peroxisomes and cytoplasm (1). Antioxidant enzymes neutralize reactive oxygen species (ROS) generated from the univalent reduction of oxygen by mitochondrial electron transport chains as well as biochemical reactions of oxygen-metabolizing enzymes (3-5). ROS, including superoxide, hydrogen peroxide, hydroxyl radical, singlet molecular oxygen, and organic hydroperoxides, are oxygen-containing molecules that have higher chemical reactivity than ground state molecular oxygen. ROS have traditionally been thought of as unwanted and toxic by-products of living in an aerobic environment (6, 7). In recent years, several studies suggest metabolic production of ROS is tightly regulated and serves a physiological function during mitogenic stimulation of cultured cells (6 -11). It has been suggested that ROS operate as a key signaling process in the cascade of events leading to cell proliferation following stimulation with platelet-derived growth factor (9), epidermal growth factor (10), cytokine...

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